1. Field of the Invention
The present disclosure relates to methods and primer sets for detecting variant(s). More particularly, the disclosed invention relates to the detection of insertion and/or deletion variant(s).
2. Description of Related Art
Gene mutations are alterations in the nucleotide sequence of a given gene or regulatory sequence from a naturally occurring or normal nucleotide sequence. A mutation may be a point mutation (single nucleotide substitution), a deletion, or insertion mutation of one or more nucleotides, a substitution mutation of more than one nucleotide, or crossing-over in chromosomal level.
Various techniques for detecting point mutations or crossing-overs have been developed. For examples, mutated nucleotide(s) could be revealed by Sanger's direct sequencing of sequence of interest. However, the sensitivity of this technique is too low, hindering its application in clinical and research uses. Alternatively, primers and/or probes specific to the target genes with point mutations or crossing-overs could be used to positively detect such mutations. However, there are few reports on detection of an insertion mutation and a deletion mutation using any of the above-mentioned method. Conventionally, primers or probes for detecting an insertion/deletion mutation are designed with prior knowledge of the sequence of the mutated site. In the case where the target gene has more than one inserted and/or deleted nucleotide, multiple primers are required to ensure the full coverage of all mutated sequences. Also, these methods tends to be error-prone, since the hybridization of the primer or probe with the target nucleic acid might occur with a mutation site being looped out, and thus unspecific hybridization occurs. Moreover, these conventional detecting methods usually have lower detection sensitivity, thereby requiring higher amount of DNA and/or more reaction cycles that are labor-intensive. In some cases, real-time quantitative polymerase chain reaction (RTQ-PCR) and/or apparatus for RTQ-PCR are required to accomplish the detection process. Therefore, in order to ensure accurate detection results, conventional methods require optimization of reaction parameters which may be costly, tedious, and/or time-consuming, and these factors limit their applications in clinical tests and basic research.
In view of the foregoing, there exists a need in the art for the development of a detection method capable of accurately detecting insertion and/or deletion mutations.